ES2970761T3 - Use of lipochitooligosaccharides and/or chitooligosaccharides in combination with phosphate-solubilizing microorganisms to enhance plant growth - Google Patents

Abstract

Disclosed are methods for enhancing plant growth, comprising a) treating plant seeds with an effective amount of at least one phosphate solubilizing microorganism that may include a strain of the fungus Penicillium, and b) treating the seed or plant germinating from the seed with an effective amount of at least one lipochitooligosaccharide (LCO) and/or at least one chitoglycosaccharide (CO), wherein upon harvest the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume, and increased leaf area, as compared to untreated plants or plants harvested from untreated seeds. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Use of lipochitooligosaccharides and/or chitooligosaccharides in combination with phosphate-solubilizing microorganisms to enhance plant growth

BACKGROUND OF THE INVENTION

The symbiosis between the gram-negative soil bacteria, Rhizobiaceae and Bradyrhizobiaceae, and legumes such as soybean is well documented. The biochemical basis of these relationships includes a molecular signaling exchange, in which plant-to-bacteria signal compounds include flavones. , isoflavones and flavanones, and bacteria-to-plant signal compounds include the end products of bradyrhizobial and rhizobial nod gene expression, known as lipochitooligosaccharides (LCO). The symbiosis between these bacteria and legumes allows the legume to fix atmospheric nitrogen for plant growth, thus avoiding the need for nitrogen fertilizers. Since nitrogen fertilizers can significantly increase the cost of crops and are associated with a number of polluting effects, the agricultural industry continues its efforts to exploit this biological relationship and develop new agents and procedures to improve plant performance without increasing the use of nitrogen fertilizers.

United States Patent No. 6,979,664 teaches a process for enhancing seed germination or seedling emergence of a plant crop, which comprises the steps of providing a composition comprising an effective amount of at least one lipochitooligosaccharide and an agriculturally suitable vehicle and applying the composition in the vicinity of a seed or seedling in an amount effective to enhance seed germination or seedling emergence compared to an untreated seed or seedling.

A further development of this concept is taught in document WO 2005/062899, which refers to combinations of at least one plant inducer, specifically an LCO, in combination with a fungicide, an insecticide or a combination thereof, to enhance a plant characteristic such as upright position, growth, vigor and/or yield of the plant. It is taught that the compositions and methods can be applied to both legumes and non-legumes and can be used to treat a seed (just before sowing), a seedling, a root or a plant.

Similarly, document WO 2008/085958 teaches compositions to enhance plant growth and crop yields in both legumes and non-legumes, which contain LCO in combination with another active ingredient such as chitin or chitosan, a flavonoid compound or a herbicide, and which can be applied to seeds and/or plants concomitantly or sequentially. As in the '899 publication, the '958 publication teaches seed treatment just before sowing.

More recently, Halford, "Smoke Signals," in Chem. Eng. News (April 12, 2010), pages 37-38, reports that karrikins or butenolides contained in smoke act as growth stimulants and stimulate germination of seeds after a forest fire, and can revitalize stored seeds such as corn, tomato, lettuce and onion. These molecules are the subject of United States Patent No. 7,576,213.

To maintain healthy growth, plants must also extract a diversity of elements from the soil in which they grow. These elements include phosphorus and so-called micronutrients (e.g., copper, iron, and zinc), but many soils are deficient in such elements or contain them only in forms that cannot be easily absorbed by plants (it is generally believed that the essential elements cannot be absorbed). be easily absorbed by plants unless present in dissolved form in the soil).

To counteract these deficiencies, sources of deficient elements are usually applied to soils to improve the growth rates and yields obtained from crop plants. For example, phosphates are often added to soil to counteract the lack of available phosphorus. Phosphate added to soil as a commercial fertilizer (e.g., monoammonium phosphate or triple superphosphate) is readily available to plants, but is quickly converted in the soil to relatively unavailable forms. It has been estimated that the plant uses only 10 to 30% of the phosphate fertilizer in the year it is applied, and the plant may never recover one-third to one-half of the phosphate fertilizer applied.

United States Patent No. 5,026,417 describes an isolated strain of Penicillium bilaiae that is capable of improving phosphorus uptake by plants when applied to soil.

Document FR 2,941,591 A1 describes the combination of chitin and tryptophan in a biofertilization process for non-legume crops.

Document WO 2012/120105 A1 describes the use of LCO derivatives and procedures to overcome the negative effects of seed treatment with fungicides, insecticides, arachicides or nematicides.

WO 2010/125065 A2 describes compositions comprising a strigolactone compound and a chitooligosaccharide (CO) to enhance plant growth and yield.

Document US 2003/096375 A1 describes procedures and compositions for the production of LCO by rhizobacteria.

However, there is still a need for systems to improve or enhance plant growth.

BRIEF SUMMARY OF THE INVENTION

A first aspect of the present invention refers to a procedure to enhance the growth of corn, which comprises:

a) treating the corn seed with an amount of 102-106 colony-forming units (cfu) per seed of at least one phosphate-solubilizing microorganism selected from the group consisting of Penicillium bilaiae strain ATCC 20851, Penicillium bilaiae strain NRRL 50169, Penicillium bilaiae strain ATCC 22348, Penicillium bilaiae strain ATCC 18309 and Penicillium bilaiae strain NRRL, and

b) treating the corn seed with an amount of about 10-5 to about 10-14 M of at least one lipochitooligosaccharide (LCO) and/or at least one chitooligosaccharide (CO), in which after harvesting the corn plant shows at least one of an increase in plant yield measured in terms of kilograms/hectare (bushels/acre), an increase in the number of roots, an increase in root length, an increase in root mass , an increase in root volume and an increase in leaf area, compared to plants harvested from untreated seeds.

Another aspect of the present invention relates to corn seeds treated with (a) at least one phosphate-solubilizing microorganism selected from the group consisting of Penicillium bilaiae strain ATCC 20851, Penicillium bilaiae strain NRRL 50169, Penicillium bilaiae strain ATCC 22348, Penicillium bilaiae strain ATCC 22348, dePenicillium bilaiaeATCC 18309 and the strain of Penicillium bilaiaeNRRL 50162 in an amount of 102-106 colony-forming units (cfu) per seed, and (b) at least one lipochitooligosaccharide (LCO) and/or at least one chitooligosaccharide (CO) in an amount from about 10-5 to about 10-14 M.

In some embodiments, the treatment of corn seed includes the direct application of the at least one phosphate-solubilizing microorganism and the at least one LCO and/or at least one CO (together "active ingredients ") on the seed, which can then be sown or stored for a period of time before sowing. Corn seed treatment may also include indirect treatment such as introducing the active ingredients into the soil (known in the art as in-furrow application). The active ingredients can be used together in a single composition or can be formulated in separate compositions for concomitant or sequential treatment. In some embodiments, corn seeds are treated with one of the active ingredients and then stored, and the other active ingredient is used to treat the corn seed at the time of planting. The methods may further include the use of other plant signal molecules and/or other agronomically beneficial agents.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 and 2 show the chemical structures of lipochitooligosaccharide (LCO) compounds useful in the practice of the present invention.

DETAILED DESCRIPTION

As used herein, "phosphate-solubilizing microorganism" is a microorganism that is capable of increasing the amount of phosphorus available to a plant.

The phosphate-solubilizing microorganism is a strain of the fungus Penicillium.

The species of Penicillium is P. bilaiae. The strains of P. bilaiaese selected from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309 and NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40:36-43).

In some embodiments, two different strains of the same species can also be combined, for example using at least two different strains of Penicillium. The use of a combination of at least two different strains of Penicillium has the following advantages. When applied to soil that already contains insoluble (or poorly soluble) phosphates, the use of combined fungal strains will result in an increase in the amount of phosphorus available for plant uptake compared to the use of a single strain of Penicillium. This, in turn, can result in an increase in phosphate uptake and/or an increase in yield of plants grown in soil compared to using individual strains alone. The combination of strains also allows insoluble rock phosphates to be used as an effective fertilizer for soils that have inadequate amounts of available phosphorus. In some embodiments, the two strains are NRRL 50169 and NRRL 50162.

Phosphate solubilizing microorganisms can be prepared using any suitable procedure known to those skilled in the art, such as solid or liquid state fermentation using a suitable carbon source. The phosphate-solubilizing microorganism is preferably prepared in the form of a stable spore.

The phosphate solubilizing microorganism is a Penicillium fungus. The Penicillium fungus according to the invention can be cultivated using solid or liquid state fermentation and a suitable carbon source. Penicillium isolates can be cultured using any suitable procedure known to those skilled in the art. For example, the fungus can be grown in a solid growth medium such as potato dextrose agar or malt extract agar, or in flasks containing suitable liquid media such as Czapek-Dox medium or potato dextrose broth. These cultivation procedures can be used in the preparation of an inoculum of Penicillium spp. for treatment (e.g., coating) of seeds and/or application to an agronomically acceptable vehicle for application to soil. The term "inoculum", as used herein, is intended to mean any form of phosphate-solubilizing microorganism (fungal cells, fungal mycelium or spores, bacterial cells or bacterial spores), which is capable of propagating on or in the soil when conditions of temperature, humidity, etc., are favorable for fungal growth.

Solid-state production of Penicillium spores, for example, can be achieved by inoculating a solid medium such as a peat or vermiculite-based substrate, or grains including, but not limited to, oats, wheat, barley or rice. The sterilized medium (obtained by autoclaving or irradiation) is inoculated with a spore suspension (1x102-1x107 cfu/ml) of the appropriate Penicillium spp. and the humidity is adjusted to 20 to 50%, depending on the substrate. The material is incubated for 2 to 8 weeks at room temperature. Spores can also be produced by liquid fermentation (Cunningham et al., 1990. Can J Bot. 68:2270-2274). Liquid production can be achieved by growing the fungus in any suitable medium, such as potato dextrose broth or sucrose yeast extract medium, under appropriate pH and temperature conditions that can be determined with standard procedures in the art.

The resulting material can be used directly, or the spores can be collected, concentrated by centrifugation, formulated and then dried using air drying, lyophilization or fluid bed drying techniques (Friesen, et al., 2005, Appl Microbiol Biotechnol 68:397- 404) to produce a wettable powder. The wettable powder is then suspended in water, applied to the surface of the corn seeds, and allowed to dry before sowing. The wettable powder may be used in conjunction with other seed treatments, such as, but not limited to, chemical seed treatments, carriers (e.g., talc, clay, kaolin, silica gel, kaolinite), or polymers (e.g., methylcellulose, polyvinylpyrrolidone). Alternatively, a spore suspension of the appropriate Penicillium spp. can be applied to a suitable soil-compatible carrier (e.g. peat-based powder or granules) until the appropriate final moisture content is reached. The material can be incubated at room temperature, typically for about 1 day to about 8 weeks, before use.

Apart from the ingredients used to cultivate the phosphate-solubilizing microorganism, including, for example, the ingredients mentioned above in the culture of Penicillium, the phosphate-solubilizing microorganism can be formulated using other agronomically acceptable vehicles. As used herein with respect to "vehicle", the term "agronomically acceptable" refers to any material that can be used to deliver the active ingredients to a corn seed, and preferably to the vehicle that can be added (to the seed) without presenting an adverse effect on plant growth, soil structure, soil drainage or the like. Suitable carriers include, but are not limited to, wheat straw, bran, ground wheat straw, peat-based powders or granules, gypsum-based granules and clays (e.g., kaolin, bentonite, montmorillonite). When adding spores to soil, a granular formulation will be preferable. Formulations such as liquid, peat or wettable powder will be suitable for coating corn seeds. When used to coat seeds, the material can be mixed with water, applied to the seeds and allowed to dry. Examples of still other vehicles include bran moistened, dried, sieved and applied to seeds previously coated with an adhesive, for example, gum arabic. In embodiments that involve the formulation of the active ingredients in a single composition, the agronomically acceptable carrier may be aqueous.

The amount of the at least one phosphate-solubilizing microorganism is effective to enhance growth such that when harvested the corn plant shows at least one of an increase in plant yield measured in terms of kilograms/hectare (bushels/hectare). acre), an increase in the number of roots, an increase in root length, an increase in root mass, an increase in root volume and an increase in leaf area, compared to plants harvested at from untreated corn seeds (with any of the active ingredients). The appropriate application rates vary depending on the type of seed or soil, the type of crop plants, the amounts of the source of phosphorus and/or micronutrients present in the soil or added to it, etc. An appropriate rate can be found through simple trial and error experiments for each particular case. Typically, for Penicillium, for example, the application rate is within the range of 0.001 to 1.0 kg of fungal spores and mycelium (fresh weight) per hectare, or 102-106 colony forming units (cfu) per seed ( when coated seeds are used), or in a granular vehicle applying between 1x106 and 1x10” colony-forming units per hectare. Fungal cells in the form of, for example, spores and the carrier can be added to a row of soil seeds at root level or used to coat the seeds before sowing, as described in more detail below. .

In embodiments, for example, involving the use of at least two strains of a phosphate-solubilizing microorganism, such as, two strains of Penicillium, according to claim 1 or 15, commercial fertilizers can be added to the soil instead of (or even additionally a) natural rock phosphate. The phosphorus source may contain a source of native soil phosphorus. In other embodiments, the phosphorus source can be added to the soil. In one embodiment, the source is rock phosphate. In another embodiment, the source is a manufactured fertilizer. Commercially available manufactured phosphate fertilizers are of many types. Some more common ones are those containing monoammonium phosphate (MAP), triple superphosphate (TSP), diammonium phosphate, ordinary superphosphate and ammonium polyphosphate. All of these fertilizers are produced by chemical processing of insoluble natural rock phosphates in large-scale fertilizer manufacturing facilities and the product is expensive. By means of the present invention it is possible to reduce the amount of these fertilizers applied to the soil while maintaining the same amount of phosphorus absorption from the soil.

In a further embodiment, the phosphorus source is organic. An organic fertilizer refers to a soil amendment derived from natural sources that guarantees at least the minimum percentages of nitrogen, phosphate and potash. Examples include plant and animal by-products, rock powders, seaweeds, inoculants and conditioners. Specific representative examples include bone meal, meat meal, animal manure, compost, sewage sludge or guano.

Of course, other fertilizers, such as nitrogen sources or other soil amendments, can also be added to the soil at around the same time as the phosphate-solubilizing microorganism or at other times, as long as the other materials are not toxic to the fungus.

Lipochitooligosaccharide compounds (LCOs), also known in the art as Nod signals or symbiotic Nod factors, consist of an oligosaccharide backbone of N-acetyl-D-glucosamine ("GlcNAc") residues linked via p-1 bonds, 4 with an N-linked fatty acyl chain fused at the non-reducing end. LCOs differ in the number of GlcNAc residues in the main chain, in the length and degree of saturation of the fatty acyl chain, and in substitutions of reducing and non-reducing sugar residues. An example of an LCO is presented below as formula I:

in which:

G is a hexosamine which may be substituted, for example, with an acetyl group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen,

R<1>, R<2>, R<3>, R<5>, R6 and R<7>, which can be the same or different, represent H, CH<3>CO--, Cx Hy CO-- wherein x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group such as for example a carbamyl,

R<4>represents a mono-, di-, or triunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

LCOs can be obtained (isolated and/or purified) from bacteria such as Rhizobia, for example, Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. The structure of LCO is characteristic of each of these bacterial species, and each strain can produce multiple LCOs with different structures. For example, in United States Patent No. 5,549,718 specific LCOs of S.melilotique have also been described as having formula II:

where R represents H or CH<3>CO-- and n is equal to 2 or 3.

Even more specific LCOs include NodRM, NodRM-1, NodRM-3. When acetylated (R=CH3 CO--), they become AcNodRM-1 and AcNodRM-3, respectively (US Patent No. 5,545,718).

LCOs from Bradyrhizobium japonicum are described in US Patent Nos. 5,175,149 and 5,321,011. Generally speaking, they are pentasaccharide phytohormones comprising methylfucose. A series of these LCOs derived fromB. japonicum: BjNod-V (C<181>); BjNod-V (Ac, C<181>), BjNod-V (C<161>), and BjNod-V (Ac, C<160>), in which "V" indicates the presence of five N-acetylglucosamines; "Ac" an acetylation; The number following the "C" indicates the number of carbons in the side chain of the fatty acid; and the number following ":" the number of double bonds.

LCOs used in embodiments of the invention can be obtained (i.e., isolated and/or purified) from LCO-producing bacterial strains, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), Sinorhizobium (including S.meliloti) and bacterial strains genetically modified to produce LCO.

LCOs are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Mol. Plant-Microbe Interactions 13:268-276 (2000)). Therefore, within the legume family, specific genera and species of rhizobia develop a symbiotic nitrogen-fixing relationship with a specific legume host. These plant-host/bacteria combinations are described by Hungria, et al., Soil Biol. Biochem.

29:819-830 (1997). Examples of these symbiotic associations between bacteria and legumes include S.meliloti/alfalfa and meliloto;R. leguminosarum biovarviciae/peas and lentils; R.leguminosarum biovar phaseoli/beans; Bradyrhizobium japonicum/soybean; and R.leguminosarum biovar trifolii/red clover. Hungary also lists the effective flavonoid Nod gene inducers of rhizobial species and the specific LCO structures that are produced by different rhizobial species. However, LCO specificity is only required to establish nodulation in legumes. In the implementation of the present invention, the use of a given LCO is not limited to the treatment of the seed of its symbiotic associated legume, in order to achieve an increase in plant yield measured in terms of kilograms/hectare ( bushels/acre), an increase in root number, an increase in root length, an increase in root mass, an increase in root volume, and an increase in leaf area, compared to harvested plants from untreated seeds, or compared to plants harvested from seeds treated with the signal molecule just before or within a period of one week or less after sowing. Thus, as an example, an LCO obtained fromB. japonicum can be used to treat seeds of legumes other than soybeans and non-legume seeds such as corn. As another example, the LCO of peas that can be obtained from R. leguminosarumillustrated in Figure 1 (designated LCO-V (C18:1), SP104) can be used to treat seeds of legumes other than peas and also seeds that are not legumes.

Also encompassed by the present invention is the use of LCOs obtained (i.e., isolated and/or purified) from mycorrhizal fungi, such as fungi of the Glomerocycota group, e.g., Glomus intraradicus. The structures of representative LCOs obtained from these fungi are described in WO 2010/049751 and WO 2010/049751 (the LCOs described therein are also called "Myc factors").

The present invention also encompasses the use of synthetic LCO compounds, such as those described in WO 2005/063784 and recombinant LCOs produced by genetic engineering. The basic structure of naturally occurring LCOs may contain modifications or substitutions found in naturally occurring LCOs, such as those described in Spaink, Crit. Rev. Plant Sci. 54:257-288 (2000) and D'Haeze, et al., Glycobiology 12:79R-105R (2002). Precursor oligosaccharide molecules (CO, which, as described below, are also useful as plant signal molecules in the present invention) for the construction of LCO can also be synthesized by genetically modified organisms, for example, such as in Samain et al., Carb. Beef.

302:35-42 (1997); Samain, et al., J. Biotechnol. 72:33-47 (1999).

LCOs can be used in various forms of purity and can be used alone or in the form of a culture of LCO-producing bacteria or fungi. For example, OPTIMIZE® (commercially available from Novozymes BioAg Limited) contains a culture ofB. japonicum which produces an LCO (LCO-V(C18:1, MeFuc), MOR116) which is illustrated in Figure 2. Methods to provide substantially pure LCO include simply removing the microbial cells from a mixture of LCO and the microbe, or continuing isolating and purifying LCO molecules by LCO solvent phase separation followed by HPLC chromatography, as described, for example, in US Patent No. 5,549,718. Purification can be improved by repeated HPLC, and the purified LCO molecules can be lyophilized for long-term storage.

Chitooligosaccharides (COs), as described above, can be used as starting materials for the production of synthetic LCOs. COs are known in the art as p-1-4 linked N-actyl-glucosamine structures identified as chitin oligomers, also as N-acetyl chitooligosaccharides. COs have unique and different side chain decorations that differentiate them from chitin molecules [(C8H13NO5)n, CAS No. 1398-61-4] and chitosan molecules [(C5HnNO4)n, CAS No. 9012-76 -4]. Representative literature describing the structure and production of CO is as follows: Van der Holst, et al., Current Opinion in Structural Biology, 11:608-616 (2001); Robina, et al., Tetrahedron 58:521-530 (2002); Hanel, et al., Planta 232:787-806 (2010); Rouge, et al. Chapter 27, "The Molecular Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 21:1053-69 (2009); PCT/F100/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120(1):83-92 (1999). Two COs suitable for use in the present invention can be easily derived from the LCOs shown in Figures 1 and 2 (less the fatty acid chains). COs can be synthetic or recombinant. Procedures for the preparation of recombinant CO are known in the art. See, for example, Samain,et al.(above); Cottaz, et al., Meth. Eng.

7(4):311 -7 (2005) and Samain, et al., J. Biotechnol. 72:33-47 (1999).

LCO and CO can be used alone or in combination. Therefore, in some embodiments, the present invention involves the use of an LCO and a CO.

Corn seeds can be treated with LCO and/or CO in various ways, such as by spraying or dripping. Spray and drip treatment can be carried out by formulating an effective amount of LCO or CO in an agriculturally acceptable carrier, usually aqueous in nature, and spraying or dripping the composition onto the seed by means of a continuous treatment system (which is calibrated to apply treatment at a predefined rate in proportion to the continuous seed flow), such as a drum type treatment device. These processes advantageously employ relatively small volumes of vehicle to allow relatively rapid drying of the treated seed. In this way large volumes of seeds can be treated effectively. Batch systems can also be employed, in which a predetermined batch size of seeds and signal molecule compositions is supplied to a mixer. Systems and apparatus to perform these processes are commercially available from numerous suppliers, for example Bayer CropScience (Gustafson).

In another embodiment, the treatment consists of coating corn seeds. One such process involves coating the interior wall of a round container with the composition, adding seeds, and then rotating the container so that the seeds come into contact with the wall and the composition, a process known in the art as "container coating." ". Seeds can be coated by combinations of coating procedures. Drenching usually involves the use of an aqueous solution containing the plant growth enhancing agent. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 h, 6 h, 12 h, 24 hours). Some types of seeds (for example, soybeans) tend to be sensitive to moisture. Therefore, it may not be desirable to soak such seeds for an extended period of time, in which case soaking is typically carried out for about 1 minute to about 20 minutes.

In those embodiments that involve storage of corn seeds after application of LCO or CO, the adhesion of the LCO or CO to the seed during any part of the storage time period is not critical. Without intending to be bound by any particular theory of operation, applicants believe that even to the extent that the treatment may not cause the plant signal molecule to remain in contact with the seed surface after treatment and during any part of storage, LCO or CO can achieve its desired effect through a phenomenon known as seed memory or seed perception. See, Macchiavelli, et al., J. Exp. Bot. 55(408):1635-40 (2004). The applicants also believe that after treatment, the LCO or CO diffuses into the young developing radicle and activates symbiotic and developmental genes, resulting in a change in the root architecture of the plant. However, to the extent desirable, compositions containing LCO or CO may also contain an adhesive or coating agent. For aesthetic purposes, the compositions may further contain a coating polymer and/or a colorant.

The amount of the at least one LCO and/or the at least one CO is effective to enhance growth such that when the corn plant is harvested it shows at least one of an increase in the yield of the plant measured in terms kilograms/hectare (bushels/acre), an increase in the number of roots, an increase in root length, an increase in root mass, an increase in root volume and an increase in leaf area, compared to plants harvested from untreated corn seeds (with any of the active ingredients). The effective amount of LCO or CO used to treat corn seed, expressed in concentration units, generally ranges from about 10-5 to about 10-14 M (molar concentration), and in some embodiments, from about 10- 5 to about 10-11 M, and in some other embodiments from about 10-7 to about 10-8 M. Expressed in units of weight, the effective amount generally ranges from about 0.02 to about 8.8 pg/ kg (from about 1 to about 400 pg/quintal (q) of seeds), and in some embodiments from about 0.04 to about 1.4 pg/kg (from about 2 to about 70 pg/q of seeds) , and in some other embodiments from about 0.05 to about 0.06 pg/kg (from about 2.5 to about 3.0 pg/q of seeds).

For indirect corn seed treatment purposes, i.e., in-furrow treatment, the effective amount of LCO or CO generally ranges from 2.5 pg/hectare (1 pg/acre) to approximately 175 pg/hectare (70 pg/acre ), and in some embodiments from about 125 pg/hectare (50 pg/acre) to about 150 pg/hectare (60 pg/acre). For purposes of application to plants, the effective amount of LCO or CO generally ranges from 2.5 pg/hectare (1 pg/acre) to about 75 pg/hectare (30 pg/acre) and, in some embodiments of approximately 27.5 pg/hectare (11 pg/acre) to approximately 50 pg/hectare (20 pg/acre).

Corn seeds can be treated with at least one phosphate-solubilizing microorganism (e.g., Penicillium) and at least one LCO and/or at least one CO just before or at the time of sowing. Treatment at the time of sowing may include direct application to the seed as described above, or in some other embodiments, by introducing the active ingredients into the soil, known in the art as treatment in the sulcus. In those embodiments that involve seed treatment followed by storage, the seeds may then be packaged, for example, in 50- or 100-pound bags, or in bulk bags or containers, according to standard techniques. The seed can be stored for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, and even longer, for example 13, 14, 15, 16, 17, 18 , 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months, or even longer, under appropriate storage conditions that are known in the art. Corn seeds can be stored for longer periods of time, even up to 3 years.

The present invention may also include treating corn seeds or plants with a plant signal molecule other than an LCO or CO. For the purposes of the present invention, the term "plant signal molecule", which may be used interchangeably with "plant growth enhancing agent", refers broadly to any agent, whether naturally occurring in plants or microbes or synthetic (and which may not be of natural origin) that directly or indirectly activates a plant biochemical pathway, resulting in an increase in plant growth, measurable at least in terms of at least one of an increase in yield measured in terms of kilograms/hectare (bushels/acre), an increase in the number of roots, an increase in root length, an increase in root mass, an increase in root volume, and an increase in leaf area , compared to plants harvested from untreated seeds. Representative examples of plant signal molecules that may be useful in practicing the present invention include chitinous compounds (other than CO), flavonoids, jasmonic acid, linoleic acid and linolenic acid and their derivatives, and karrikins.

Chitins and chitosans, which are major components of the cell walls of fungi and the exoskeletons of insects and crustaceans, are also composed of GlcNAc residues. Chitinous compounds include chitin, (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2yl]methoxymethyl]-2-[[5-acetylamino-4,6- dihydroxy-2-(hydroxymethyl)oxan-3-yl]methoxymethyl]-4-hydroxy-6-(hydroxymethyl)oxan-3-is]ethanamide) and chitosan (IUPAC: 5-amino-6-[5-amino -6 -[5-amino-4,6-dihydroxy-2(hydroxymethyl)oxan-3-yl]oxy-4-hydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-2(hydroxymethyl)oxane-3,4 -diol). These compounds can be obtained commercially, for example, from Sigma-Aldrich, or prepared from insects, crustacean shells or fungal cell walls. Procedures for the preparation of chitin and chitosan are known in the art and have been described, for example, in United States Patent No. 4,536,207 (preparation from crustacean shells), Pochanavanich, et al., Lett . Appl. Microbiol. 35:17-21 (2002) (preparation from fungal cell walls) and US Patent No. 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans can be obtained that range from less than 35% to more than 90% deacetylation, and span a wide spectrum of molecular weights, for example, low molecular weight chitosan oligomers of less than 15 kD and chitin oligomers. from 0.5 to 2 kD; "practical grade" chitosan with a molecular weight of approximately 15 kD; and high molecular weight chitosan of up to 70 kD. Chitin and chitosan compositions formulated for seed treatment are also commercially available. Commercial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYOND™ (Agrihouse, Inc.).

Flavonoids are phenolic compounds that have the general structure of two aromatic rings connected by a three-carbon bridge. Flavonoids are produced by plants and have many functions, for example as beneficial signaling molecules and as protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See, Jain, et al., J. Plant Biochem. & Biotechnol. 11:1-10 (2002); Shaw, et al., Environmental Microbiol.

11:1867-80 (2006).

Representative flavonoids that may be useful in practicing the present invention include genistein, daidzein, formononetin, naringenin, hesperetin, luteolin and apigenin. Flavonoid compounds are commercially available, for example, from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds can be isolated from plants or seeds, for example, as described in US Patent Nos. 5,702,752, 5,990,291 and 6,146,668. Flavonoid compounds can also be produced by genetically engineered organisms, such as yeast, as described in Ralston, et al., Plant Physiology 137:1375-88 (2005).

Jasmonic acid (JA, [1 R-[1a,2p(Z)]]-3-oxo-2-(pentenyl)cyclopentaneacetic acid) and its derivatives, linoleic acid ( (Z,Z)-9,12-octadecadienoic acid) and its derivatives, and linolenic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid) and its derivatives. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid-based compounds found naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings and by fungal microorganisms such as Botryodiplodia theobroma and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Linoleic acid and linolenic acid are produced during the biosynthesis of jasmonic acid. Jasmonates, linoleic acid, and linoleic acid (and their derivatives) are reported to be inducers of nod gene expression or LCO production by rhizobacteria. See, for example, Mabood, Fazli, Jasmonates induces the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induces the expression of nod genes in Bradyrhizobium japonicum", USDA 3, May 17, 2001.

Useful derivatives of linoleic acid, linolenic acid and jasmonic acid that may be useful in practicing the present invention include esters, amides, glycosides and salts. Representative esters are compounds in which the carboxyl group of linoleic acid, linolenic acid or jasmonic acid has been replaced by a -COR group, where R is a --OR1 group, where R1 is: a group alkyl, such as a branched or unbranched C<1>-C<8>alkyl group, for example, a methyl, ethyl or propyl group; an alkenyl group, such as a branched or unbranched C<2>-C<8>alkenyl group; an alkynyl group, such as a branched or unbranched C<2>-C<8>alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, the heteroatoms in the heteroaryl group may be, for example, N, O, P or S. Representative amides are compounds in which the carboxyl group of the linoleic acid, linolenic acid or jasmonic acid has been replaced by a --COR group, where R is an NR2R3 group, where R2 and R3 are independently: hydrogen; an alkyl group, such as a C<1>-C<8>branched or unbranched alkyl group, for example, a methyl, ethyl or propyl group; an alkenyl group, such as a branched or unbranched C<2>-C<8>alkenyl group; an alkynyl group, such as a branched or unbranched C<2>-C<8>alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, the heteroatoms in the heteroaryl group may be, for example, N, O, P or S. The esters can be prepared by known procedures, such as addition acid-catalyzed nucleophilic, in which the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid. Amides can also be prepared by known procedures, such as reacting the carboxylic acid with the appropriate amine in the presence of a coupling agent such as dicyclohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of linoleic acid, linolenic acid and jasmonic acid include, for example, base addition salts. Bases that can be used as reagents to prepare metabolically acceptable base salts of these compounds include those derived from cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts can be easily prepared by mixing together a solution of linoleic acid, linolenic acid or jasmonic acid with a solution of the base. The salt may be precipitated from solution and collected by filtration or may be recovered by other means such as by evaporation of the solvent.

Karrikins are vinylogue 4H-pyrones, for example, 2H-furo[2,3-c]pyran-2-ones, including derivatives and analogues thereof. Examples of these compounds are represented by the following structure:

in which; Z is O, S or NRs; Ri, R<2>, R<3>and R<4>are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, OR6, COOR =, halogen, NR<6>R<7>or NO<2>; and R<5>, R6 and R<7>are each independently H, alkyl or alkenyl, or a biologically acceptable salt thereof. Examples of biologically acceptable salts of these compounds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate , citrate, tartrate, gluconate; methanesulfonate, benzenesulfonate and p-toluenesulfonic acid. Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include sodium and potassium salts. Examples of compounds covered by the structure and which may be suitable for use in the present invention include the following: 3-methyl-2H-furo[2,3-c]pyran-2-one (wherein R1=CH3 , R<2>, R<3>, R4=H), 2H-furo[2,3-c]pyran-2-one (in which R<1>, R<2>, R<3>, R4=H), 7-methyl-2H-furo[2,3-c]pyran-2-one (where R<1>, R<2>, R4=H, R3=CH3), 5-methyl -2H-furo[2,3-c]pyran-2-one (where R<1>, R<2>, R<3>=H, R4=CH3), 3,7-dimethyl-2H- furo[2,3-c]pyran-2-one (where R<1>, R3=CH3, R<2>, R<4>=H), 3,5-dimethyl-2H-furo[2 ,3-c]pyran-2-one (where R<1>, R4=CH3, R<2>, R<3>=H), 3,5,7-trimethyl-2H-furo[2, 3-c]pyran-2-one (where R<1>, R<3>, R4=CH3, R<2>=H), 5-methoxymethyl-3-methyl-2H-furo[2,3 -c]pyran-2-one (where R1=CH3, R<2>, R<3>=H, R4=CH2OCH3), 4-bromo-3,7-dimethyl-2H-furo[2,3 -c]pyran-2-one (in which R<1>, R3=CH3, R<2>=Br, R4=H), 3-methylfuro[2,3-c]pyridin-2(3H)- one (in which Z=NH, R1=CH3, R<2>, R<3>, R4=H), 3,6-dimethylfuro[2,3-c]pyridin-2(6H)-one (in which Z=N--CH3, R1=CH3, R<2>, R<3>, R4=H). See US Patent No. 7,576,213. These molecules are also known as karrikins. See Halford, above.

The present invention may further include treating corn seed with an agriculturally/agronomically beneficial agent. As used herein and in the art, the term "agriculturally or agronomically beneficial" refers to agents that when applied to seeds result in an improvement (which may be statistically significant) of plant characteristics such as such as upright position, growth, vigor or yield of the plant compared to untreated seeds. Representative examples of such agents that may be useful in practicing the present invention include herbicides, fungicides and insecticides.

Suitable herbicides include bentazone, acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, setoxydim, imazethapyr, imazamox, fomesafo, flumichlorac, imazaquin and clethodim. Commercial products containing each of these compounds are readily available. The concentration of herbicide in the composition will generally correspond to the indicated use rate for a particular herbicide.

A "fungicide", as used herein and in the art, is an agent that kills or inhibits the growth of fungi. As used herein, a fungicide "exhibits activity against" a particular species of fungus if treatment with the fungicide results in the death or inhibition of growth of a fungal population (e.g., in soil). with respect to an untreated population. Effective fungicides according to the invention will suitably show activity against a wide range of pathogens, including, but not limited to, Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora and combinations thereof.

Commercial fungicides may be suitable for use in the present invention. Suitable commercially available fungicides include PROTÉGÉ, RIVAL or ALLEGIAn Ce FL or LS (Gustafson, Plano, TX), WARDEN RTA (Agrilance, St. Paul, MN), APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta , Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, Argentina). The active ingredients of these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most appropriately used according to the manufacturer's instructions at recommended concentrations.

As used herein, an insecticide "exhibits activity against" a particular species of insect if treatment with the insecticide results in the killing or inhibition of an insect population relative to an untreated population. Effective insecticides according to the invention will suitably show activity against a wide range of insects including, but not limited to, wireworms, cutworms, larvae, corn rootworms, corn seedworms, flea beetles, stink bugs, aphids, leaf beetles and stink bugs.

Commercial insecticides may be suitable for use in the present invention. Suitable commercially available insecticides include CRUISER (Syngenta, Wilmington, DE), GAUCHO, and PONCHO (Gustafson, Plano, TX). The active ingredients of these and other commercial insecticides include thiamethoxam, clothianidin and imidacloprid. Commercial insecticides are most appropriately used according to the manufacturer's instructions at recommended concentrations.

The procedures of the present invention are applicable to corn seeds.

All patent and non-patent publications cited herein are indicative of the level of skill of those skilled in the art to which the present invention pertains.

Claims (15)

1. A procedure to enhance the growth of corn, which includes a) treating a corn seed with an amount of 102-106 colony-forming units (cfu) per seed of at least one phosphate-solubilizing microorganism selected from the group consisting of Penicillium bilaiae strain ATCC 20851, Penicillium bilaiae strain NRRL 50169, strain of Penicillium bilaiae ATCC 22348, the strain of Penicillium bilaiae ATCC 18309 and the strain of Penicillium bilaiae Nr Rl 50162, and b) treating the corn seed that germinates from the seed with an amount of about 10-5 to about 10-14 M of at least one lipochitooligosaccharide (LCO) and/or at least one chitooligosaccharide (CO), wherein After harvest the corn plant shows at least one of an increase in plant yield measured in terms of kilograms/hectare (bushels/acre), an increase in the number of roots, an increase in root length, a increased root mass, an increase in root volume and an increase in leaf area, compared to plants harvested from untreated seeds. 2. The method of claim 1, wherein the at least one phosphate-solubilizing microorganism and the at least one LCO and/or at least one CO are applied to the seeds before or about sowing. at the time of sowing. 3. The method of claim 1, wherein the at least one phosphate-solubilizing microorganism and the at least one LCO and/or at least one CO are applied to the seeds in the furrow. 4. The method of claim 1, wherein the at least one phosphate-solubilizing microorganism and the at least one LCO and/or at least one CO are applied to the seeds by means of a single composition. . 5. The method of claim 1, wherein the at least one phosphate-solubilizing microorganism and the at least one LCO and/or at least one CO are applied to the seeds by means of different compositions. 6. The method of claim 1, further comprising applying to the seed at least one plant signal molecule other than an LCO or CO. 7. The method of claim 6, wherein the plant signal molecule is a chitinous compound. 8. The method of claim 6, wherein the plant signal molecule is a flavonoid. 9. The method of claim 6, wherein the plant signal molecule is jasmonic acid or a derivative thereof. 10. The method of claim 6, wherein the plant signal molecule is linoleic acid or a derivative thereof. 11. The method of claim 6, wherein the plant signal molecule is linolenic acid or a derivative thereof. 12. The method of claim 6, wherein the plant signal molecule is a karrikin. 13. The method of claim 8, wherein the flavonoid is selected from the group consisting of genistein, daidzein, formononetin, naringenin, hesperetin, luteolin and apigenin. 14. The method of claim 1 to 13, further comprising applying to the plant or seeds thereof at least one agronomically beneficial agent, wherein the agronomically beneficial agent is a herbicide, an insecticide, a fungicide or any combination thereof. 15. Corn seed that has been coated with, or on which has been arranged, (a) at least one phosphate-solubilizing microorganism selected from the group consisting of Penicillium bilaiae ATCC 20851 strain, Penicillium bilaiae strain NRRL 50169, Penicillium bilaiae strain bilaiaeATCC 22348, Penicillium bilaiae ATCC 18309 strain and Penicillium bilaiae NRRL 50162 strain in an amount of 102-106 colony forming units (cfu) per seed, and (b) at least one lipochitooligosaccharide (LCO) and/or at least one chitooligosaccharide ( CO) in an amount of about 10-5 to about 10-14 M.